Chimeric receptor for improving killing activity of immune cells and application thereof

Abstract

The present invention belongs to the field of biological medicines, and particularly relates to a chimeric receptor for improving the killing activity of immune cells and an application thereof. Specifically, the present invention provides a fusion protein. The fusion protein comprises an extracellular part, an extracellular hinge region, a transmembrane region, and an intracellular region. Most preferably, the amino acid sequence of the fusion protein of the present invention is formed by sequentially connecting SEQ ID NO.: 1, SEQ ID NO.: 3, SEQ ID NO.: 5, SEQ ID NO.: 7, SEQ ID NO.: 9 and SEQ ID NO.: 11, and the immune cells expressing the fusion protein have strong killing activity.

1 . A fusion protein, comprising an extracellular portion, an extracellular hinge region, a transmembrane region, and an intracellular portion, wherein the extracellular portion is any of the following:
 1. Ig-like C2 type 1, Ig-like C2 type 2 of CD16a and Ig-like C2 type 3 of CD64 are sequentially connected in series;   2. Ig-like C2 type 1 of CD16A, Ig-like C2 type 2 of CD16A, Ig-like C2 type 1 of CD64A, Ig-like C2 type 2 of CD64A, and Ig-like C2 type 3 of CD64A are sequentially connected in series;   3. Ig-like C2 type 1 of CD64A, Ig-like C2 type 2 of CD64A, Ig-like C2 type 3 of CD64A, Ig-like C2 type 1 of CD16A, and Ig-like C2 type 2 of CD16A are sequentially connected in series;   4. Ig-like C2 type 1 of CD64A, Ig-like C2 type 2 of CD64A, Ig-like C2 type 3 of CD64A, and Ig-like C2 type 1 of CD16A are sequentially connected in series; and   5. Ig-like C2 type 1 of CD64A, Ig-like C2 type 2 of CD64A, Ig-like C2 type 3 of CD64A, and Ig-like C2 type 2 of CD16A are sequentially connected in series; and   wherein the extracellular hinge region, the transmembrane region, and the intracellular region are respectively the extracellular hinge region of CD64, the transmembrane region of CD16a, and the intracellular region of CD16a.   
     
     
         2 . The fusion protein as claimed in  claim 1 , wherein:
 the amino acid sequence of Ig-like C2 type 1 of CD16a is shown in SEQ ID NO.: 1 or has 1, 2, 3, 4, 5 or more mutations compared to the sequence shown,   the amino acid sequence of Ig-like C2 type 2 of CD16a is shown in SEQ ID NO.: 3 or has 1, 2, 3, 4, 5 or more mutations compared to the sequence shown,   the amino acid sequence of the Ig-like C2 type 3 of CD64 is shown in SEQ ID NO.: 5 or has 1, 2, 3, 4, 5 or more mutations compared to the sequence shown,   the amino acid sequence of the extracellular hinge region of CD64 is shown in SEQ ID NO.: 7 or has 1, 2, 3, 4, 5 or more mutations compared to the sequence shown,   the amino acid sequence of the transmembrane region of CD16a is shown in SEQ ID NO.: 9 or has 1, 2, 3, 4, 5 or more mutations compared to the sequence shown,   the amino acid sequence of the intracellular region of CD16a is shown in SEQ ID NO.: 11 or has 1, 2, 3, 4, 5 or more mutations compared to the shown sequence,   the amino acid sequence of the intracellular region of CD64A Ig-like C2 type 1 is shown in SEQ ID NO.: 15 or has 1, 2, 3, 4, 5, or more mutations with the shown sequence, and   the amino acid sequence of the intracellular region of CD64A Ig-like C2 type 2 is shown in SEQ ID NO.: 17 or has 1, 2, 3, 4, 5 or more mutations with the shown sequence.   
     
     
         3 . The fusion protein as claimed in  claim 1 , wherein the fusion protein is coded by a coding nucleic acid. 
     
     
         4 . The fusion protein as claimed in  claim 3 , wherein:
 the encoding nucleic acid sequence of Ig-like C2 type 1 of CD16a has 85% or more homology with the sequence shown in SEQ ID NO.: 2, partially or completely complementary with the sequence shown in SEQ ID NO.: 2, or as shown in SEQ ID NO.: 2,   the coding nucleic acid sequence of Ig-like C2 type 2 of CD16a has 85% or more homology with the sequence shown in SEQ ID NO.: 4, or partially or completely complementary with the sequence shown in SEQ ID NO.: 4, or as shown in SEQ ID NO.: 4,   the coding nucleic acid sequence of Ig-like C2 type 3 of CD64 has 85% or more homology with the sequence shown in SEQ ID NO.: 6, or partially or completely complementary with the sequence shown in SEQ ID NO.: 6, or as shown in SEQ ID NO.: 6,   the coding nucleic acid sequence of the extracellular hinge region has 85% or more homology with the sequence shown in SEQ ID NO.: 8, or partially or completely complementary with the sequence shown in SEQ ID NO.: 8, or as shown in SEQ ID NO.: 8,   the coding nucleic acid sequence of the transmembrane region has 85% or more homology with the sequence shown in SEQ ID NO.: 10, or partially or completely complementary with the sequence shown in SEQ ID NO.: 10, or as shown in SEQ ID NO.: 10,   the coding nucleic acid sequence of the intracellular region has 85% or more homology with the sequence shown in SEQ ID NO.: 12, or partially or completely complementary with the sequence shown in SEQ ID NO.: 12, or as shown in SEQ ID NO.: 12,   the coding nucleic acid sequence of CD64A Ig-like C2 type 1 has 85% or more homology with the sequence shown in SEQ ID NO.: 16, or partially or completely complementary with the sequence shown in SEQ ID NO.: 16, or as shown in SEQ ID NO.: 16, and   the coding nucleic acid sequence of CD64A Ig-like C2 type2 has 85% or more homology with the sequence shown in SEQ ID NO.: 18, or partially or completely complementary with the sequence shown in SEQ ID NO.: 18, or as shown in SEQ ID NO.: 18.   
     
     
         5 . The fusion protein as claimed in  claim 1 , wherein the fusion protein is expressed by an expression vector or a host cell. 
     
     
         6 . The fusion protein as claimed in  claim 5 , wherein the expression vector includes but is not limited to bacterial plasmid vector, bacteriophage vector, yeast plasmid vector, adenovirus vector, retrovirus vector, and lentivirus vector. 
     
     
         7 . The fusion protein as claimed in  claim 5 , wherein the host cell includes human immune cells or stem cells. 
     
     
         8 . The fusion protein as claimed in  claim 7 , wherein the immune cells include one or more of T cells, B cells, K cells, and NK cells. 
     
     
         9 . The fusion protein as claimed in  claim 7 , wherein the immune cell is NK cell and the stem cell is iPSC. 
     
     
         10 . A method for preparing immune cells with high cytotoxicity, comprising introducing one or more of the fusion protein, the polynucleotide, and the vector into immune cells;
 alternatively, the method comprises introducing one or more of the fusion protein, the polynucleotide, and the vector into stem cells, and then inducing stem cells to differentiate into immune cells.   
     
     
         11 . The method as claimed in  claim 10 , wherein the method includes electroporation, protoplast fusion, calcium phosphate precipitation, cell fusion using encapsulated DNA, microinjection, and complete virus transfection. 
     
     
         12 . The method as claimed in  claim 10 , further comprising contacting immune cells with cancer cells in vitro. 
     
     
         13 . The method as claimed in  claim 12 , wherein the cancer cells are prostate cancer cells. 
     
     
         14 . An application comprising:
 the application of one or more of the fusion protein, the polynucleotide, the vector, and host cell as a pharmaceutical composition;   or, the application of one or more of the fusion protein, the polynucleotide, the vector, the host cell and the pharmaceutical composition in preparation of cancer immunotherapy drugs, autoimmune disease drugs, anti-aging drugs, medical beauty products, and metabolic disease drugs;   or the application of one or more of the fusion protein, the polynucleotide, the vector, the host cell and the pharmaceutical composition in improving the therapeutic effect of monoclonal antibodies.   
     
     
         15 . The application as claimed in  claim 14 , wherein the pharmaceutical composition also contains other drugs for treating cancer and/or structures recognized by the fusion protein. 
     
     
         16 . The application as claimed in  claim 14 , wherein the drug is a monoclonal antibody drug, which includes an antibody with the product number ab268061 provided by abcam company or a marketed monoclonal antibody drug, the marketed monoclonal antibody drug includes Matuximab, Trastuzumab, Cituximab, Dalizumab, Tanizumab, Abavozumab Admuzumab, Aftuzumab, Alenzumab, Peihua Aczumab, Almatuzumab, Abazumab, Paviximab, Betomozumab, Belimuzumab, Bevaczumab, Mobivaczumab, Berentzumab Vititin, Mocantzumab, Lacantzumab, Carolizumab Penditide, Carotozumab, Positazumab, Situxumab, Konamizumab, Dacitazumab Dalozumab, Desmozumab, Emexizumab, Ezuzuzumab, Ezuzuzumab, Ensiximab, Epacizumab, Emasozumab, Adazumab, Falezumab, Fentolumab, Galicizumab, Gizuzumab, Gizuzumab, Giriximab, Gleizumab Vititin, Teimozumab, Igovozumab, Laindacizumab, Intuxumab, Izumab, Ozomicin Epilimumab, Itomumab, Labezumab, Laishamumab, Lintuzumab, or Molovozumab. 
     
     
         17 . The application as claimed in  claim 14 , wherein:
 the pharmaceutical composition further comprises pharmaceutically acceptable carriers, diluents, or excipients,   the pharmaceutically acceptable carriers, diluents, or excipients include, but are not limited to, any adjuvants, vectors, excipients, flow aids, sweeteners, diluents, anti-corrosion agents, dyes/colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspensions, stabilizers, isotonic agents, solvents that have been approved by the US Food and Drug Administration or the China Food and Drug Administration for use in humans or livestock Surfactants or emulsifiers, and   the pharmaceutical composition is tablets, pills, powders, granules, capsules, pastilles, syrups, liquids, emulsions, suspensions, controlled release preparations, aerosols, films, injections, intravenous drops, transdermal absorption preparations, ointments, lotions, adhesive preparations, or suppositories.   
     
     
         18 . The application as claimed in  claim 14 , wherein:
 the cancers include cervical cancer, seminoma, testicular lymphoma, prostate cancer, ovarian cancer, lung cancer, rectal cancer, breast cancer, skin squamous cell cancer, colon cancer, liver cancer, pancreatic cancer, stomach cancer, esophageal cancer, thyroid cancer, bladder transitional epithelial cancer, leukemia, brain tumor, stomach cancer, peritoneal cancer, head and neck cancer, endometrial cancer, kidney cancer Female reproductive tract cancer, in situ cancer, neurofibroma, bone cancer, skin cancer, gastrointestinal stromal tumor, mast cell tumor, multiple myeloma, melanoma, or glioma,   the autoimmune disease includes achalasia, Addison's disease, adult Steele's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti GBM/anti TBM nephritis, antiphospholipid syndrome, autoimmune vascular edema, autoimmune autonomic dysfunction, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease, autoimmune myocarditis Autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, or autoimmune urticaria, and the metabolic diseases include diabetes, diabetes ketoacidosis, hyperglycemia and hypertonic syndrome, hypoglycemia, gout, protein energy malnutrition, vitamin A deficiency, scurvy, vitamin D deficiency or osteoporosis.   
     
     
         19 . The application as claimed in  claim 14 , wherein the therapeutic effect is specific to prostate cancer.